1. Field of the Invention
This invention relates to a process for secretory production of a biogenic substance, which process is characterized by fusing useful proteins with a calcium-binding protein
2. Description of the Related Art
Photoprotein aequorin is a calcium-binding protein isolated from photogenic Aequorea living in the ocean near the suburbs of Friday Harbor Island, Washington State, U.S.A. As to the aequorin, apoaequorin as a protein portion and coelenterazine as a substrate portion form a complex in a medium containing molecular oxygen in nature, and the aequorin is characterized by causing luminescence by binding of calcium with the complex. By utilizing this luminescence, it is possible to measure calcium concentration.
The present inventors cloned cDNA of apoaequorin from photogenic Aequorea according to recombinant DNA technique and determined its primary structure (Japanese patent application laid-open No. Sho 61-135,586), and then using this cDNA and Escherichia coli as host, succeeded in intracellular and extracellular production of apoaequorin (Japanese patent application Nos. Sho 60-280,259 and Sho 61-249,098). Further, we prepared aequorin gene bound with the functional gene and succeeded in production of a fusion protein thereof (Japanese patent application No. Sho 62-196,031). Further, we developed a metal-detecting method by the use of luminescence of aequorin (Japanese patent application No. Sho 61-103,849). Further, we prepared aequorin gene bound with the gene of specifically bound protein and succeeded in production of fusion protein thereof (Japanese patent application No. Sho 63-308,424).
Further, in order to utilize the fusion protein for immunoassay of enzyme, we established a method for preparing a high purity specimen of the fusion protein (Japanese patent application No. Hei 1-69,862), and succeeded in practical application thereof to immunoassay (Japanese patent application No. Hei 1-74,742).
The present invention is based upon analysis of the mechanism of extracellular secretory production of apoaequorin in cells using Escherichia coli as host, and is directed to production of a useful biogenic substance utilizing the secretion production system.
Now, the usefulness of aequorin has been well known to persons skilled in the art, and by utilizing the luminescence of aequorin, it is possible to detect various substances. Namely, aequorin is applicable to all measurement systems such as immunoassay, DNA probe, biosensor, etc., and in view of the above-mentioned functions, aequorin can be expected to be useful as agents such as used in diagnostics.
In view of the above-mentioned technical situations, the present inventors have made extensive research, and as a result, have elucidated the mechanism of the secretory production of apoaequorin using Escherichia coli as host, and could have invented a process for secretory production of a useful biogenic substance.